ABSTRACT
The extracellular crude dextransucrase (0.67 U/mg) from P. pentosaceus CRAG3 (GenBank accession number JX679020) after PEG-1500 fractionation gave specific activity, 20.0 U/mg which by gel filtration resulted in 46.0 U/mg. The purified dextransucrase displayed molecular size of approximately, 224 kDa. The optimum assay conditions for dextransucrase activity were 5% sucrose in 20 mM sodium acetate buffer (pH 5.4) and 30 oC. The dextransucrase was stable up to 40 oC and at pH range of 5.4-7.0. The metal ions such as Co2+, Ca2+, Mg2+ and Zn2+ stimulated the dextransucrase activity by 56, 44, 14 and 12%, respectively. It was most stable at -20 oC with half-life of 307 days. Amongst various additives used, glycerol and Tween 80 provided significant stability to the enzyme with half-life 15.5 and 85.5 h, respectively as compared to control (6.9 h). The solidification of sucrose supplemented milk by purified dextransucrase due to dextran synthesis displayed its application as additive for improving the texture of dairy products.
Subject(s)
Cations, Divalent/pharmacology , Chromatography, Gel , Drug Storage , Electrophoresis, Polyacrylamide Gel , Food Additives , Fungal Proteins/chemistry , Fungal Proteins/isolation & purification , Glucosyltransferases/chemistry , Glucosyltransferases/isolation & purification , Half-Life , Hydrogen-Ion Concentration , Molecular Weight , Pediococcus/enzymology , Protein Stability , TemperatureABSTRACT
The mutant of Pediococcus pentosaceus (SPAm) produced earlier by UV-mutagenesis exhibiting higher dextransucrase activity as compared to wild-type was used. The generated mutant SPAm gave 12.2 mg/ml, a 20 percent higher dextran than wild-type. Response surface methodology was carried out for further enhancement of dextran production. To enhance dextran production by the mutant SPAm, Plackett-Burman Design and a 2² full factorial Central Composite Design was employed. After response optimization, the optimum concentration of sucrose and yeast extract was 5.115 percent (w/v) and 0.635 percent (w/v), respectively. The experimental values of dextran 36.0 mg/ml at flask level and 35.0 mg/ml at bioreactor level were in good agreement with the predicted value of 40.8 mg/ml. The increase in dextran production by the mutant SPAm using the optimized medium was 3 fold higher as compared to unoptimized medium.